The invention provides a new process for the fermentative preparation of L-threonine with Enterobacteriaceae.
L-Threonine is used in animal nutrition, in human medicine and in the pharmaceuticals industry.
It is known that L-threonine can be prepared by fermentation of strains of the Enterobacteriaceae family, in particular Escherichia coli. Because of the great importance of this amino acid, work is constantly being undertaken to improve the preparation processes. Improvements to the process can relate to fermentation measures, such as e.g. stirring and supply of oxygen, or the composition of the nutrient media, such as e.g. the sugar concentration during the fermentation, or the working up to the product form, by e.g. ion exchange chromatography, or the intrinsic output properties, i.e. those of genetic origin, of the microorganism itself.
It is known from the prior art, such as is described, for example, in U.S. Pat. No. 5,538,873 and in EP-B-0593792 or by Okamoto et al. (Bioscience, Biotechnology, and Biochemistry 61 (11), 1877xe2x80x941882, 1997), that threonine is prepared by fermentation in the batch process (batch) or feed process (fed batch).
The inventors had the object of providing new measures for improved fermentative preparation of L-threonine.
The invention provides a fermentation process, which is characterized in that
a) an L-threonine-producing microorganism of the Enterobacteriaceae family is cultured by the feed process (fed batch) in a known manner, subsequently
b) a portion of the fermentation broth is separated off, 1 to 90 vol. %, in particular 1 to 50 vol. %, preferably 1 to 25 vol. % and particularly preferably 5 to 50 vol. % of the total volume of the fermentation broth remaining in the fermentation tank, subsequently
c) the remaining fermentation broth is topped up with growth medium and, preferably after a growth phase, a further fermentation is carried out by the feed process (fed batch) mentioned,
d) steps b) and c) are optionally carried out several times, and
e) the L-threonine is isolated from the fermentation broths collected.
The microorganisms with which the process according to the invention can be carried out can prepare L-threonine from glucose, sucrose, lactose, fructose, maltose, molasses, starch, or from glycerol and ethanol, the preparation from glucose, sucrose or molasses being preferred. They are representatives of Enterobacteriaceae, in particular of the genera Escherichia, Serratia and Providencia. Of the genus Escherichia the species Escherichia coli and of the genus Serratia the species Serratia marcescens are to be mentioned in particular.
Suitable L-threonine-producing strains of the genus Escherichia, in particular of the species Escherichia coli , are, for example
Escherichia coli TF427
Escherichia coli H-4225
Escherichia coli H-4226
Escherichia coli H-4257
Escherichia coli H-4258
Escherichia coli H-4435
Escherichia coli H-4436
Escherichia coli H-4578
Escherichia coli H-7256
Escherichia coli H-7263
Escherichia coli H-7293
Escherichia coli H-7294
Escherichia coli H-7700
Escherichia coli H-7729
Escherichia coli H-8309
Escherichia coli H-8311
Escherichia coli H-9244
Escherichia coli KY10935
Escherichia coli EL1003
Escherichia coli VNIIgenetika MG-442
Escherichia coli VNIIgenetika VL334/pYN7
Escherichia coli VNIIgenetika M1
Escherichia coli VNIIgenetika 472T23
Escherichia coli VNIIgenetika TDH-
Escherichia coli BKIIM B-3996
Escherichia coli BKIIM B-5318
Escherichia coli B-3996-C43
Escherichia coli B-3996-C80
Escherichia coli B-3996/pTWV-pps
Escherichia coli B-3996(pMW::THY)
Escherichia coli B-3996/pBP5
Escherichia coli Ferm BP-3756
Escherichia coli Ferm BP-4072
Escherichia coli Ferm BP-1411
Escherichia coli kat 13
Escherichia coli KCCM-10132
Escherichia coli KCCM-10133.
Suitable L-threonine-producing strains of the genus Serratia, in particular of the species Serratia marcescens, are, for example
Serratia marcescens HNr21
Serratia marcescens TLr156
Serratia marcescens T2000
Strains from the Enterobacteriaceae family which produce L-threonine preferably have, inter alia, one or more genetic or phenotypic features chosen from the group consisting of: resistance to xcex1-amino-xcex2-hydroxyvaleric acid, resistance to thialysine, resistance to ethionine, resistance to xcex1-methylserine, resistance to diaminosuccinic acid, resistance to a-aminobutyric acid, resistance to borrelidin, resistance to rifampicin, resistance to valine analogues, such as, for example, valine hydroxamate, resistance to purine analogues, such as, for example, 6-dimethylaminopurine, a need for L-methionine, optionally a partial and compensatable need for L-isoleucine, a need for meso-diaminopimelic acid, auxotrophy in respect of threonine-containing dipeptides, resistance to L-threonine, resistance to L-homoserine, resistance to L-lysine, resistance to L-methionine, resistance to L-glutamic acid, resistance to L-aspartate, resistance to L-leucine, resistance to L-phenylalanine, resistance to L-serine, resistance to L-cysteine, resistance to L-valine, sensitivity to fluoropyruvate, defective threonine dehydrogenase, optionally an ability for sucrose utilization, enhancement of the threonine operon, enhancement of homoserine dehydrogenase I-aspartate kinase I, preferably of the feed back resistant form, enhancement of homoserine kinase, enhancement of threonine synthase, enhancement of aspartate kinase, optionally of the feed back resistant form, enhancement of aspartate semialdehyde dehydrogenase, enhancement of phosphoenol pyruvate carboxylase, optionally of the feed back resistant form, enhancement of phosphoenol pyruvate synthase, enhancement of transhydrogenase, enhancement of the RhtB gene product, enhancement of the RhtC gene product, enhancement of the YfiK gene product, enhancement of a pyruvate carboxylase, and attenuation of acetic acid formation.
Thus, for example, the strain 472T23 (U.S Pat. No. 5,631,157) has, inter alia, an enhanced, xe2x80x9cfeed backxe2x80x9d resistant aspartate kinase I-homoserine dehydrogenase I, an attenuated threonine deaminase, a resistance to at least 5 g/l L-threonine and the ability to utilize sucrose as a source of carbon.
Thus, for example, the strain B-3996 (U.S. Pat. No. 5,175,107) has, inter alia, an enhanced, xe2x80x9cfeed backxe2x80x9d resistant aspartate kinase I-homoserine dehydrogenase I, an attenuated threonine deaminase, an attenuated threonine dehydrogenase, a resistance to at least 5 g/l L-threonine and the ability to utilize sucrose as a source of carbon.
Thus, for example, the strain kat-13 (U.S. Pat. No. 5,939,307) has, inter alia, an enhanced, xe2x80x9cfeed backxe2x80x9d resistant aspartate kinase I-homoserine dehydrogenase I, an attenuated threonine dehydrogenase, resistance to borrelidin and the ability to utilize sucrose as a source of carbon.
Thus, for example, the strain KCCM-10132 (WO 00/09660) has a resistance to xcex1-methylserine, a resistance to diaminosuccinic acid, sensitivity to fluoropyruvate, a resistance to L-glutamic acid and a resistance to at least 7% L-threonine. The strain is also in need of the amino acids L-methionine and L-isoleucine.
The term xe2x80x9cenhancementxe2x80x9d in this connection describes the increase in the intracellular activity of one or more enzymes in a microorganism which are coded by the corresponding DNA, for example by increasing the number of copies of the gene or allele or of the genes or alleles, using a potent promoter or using a gene or allele which codes for a corresponding enzyme having a high activity, and optionally combining these measures.
By enhancement measures, in particular over-expression, the activity or concentration of the corresponding protein is in general increased by at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, up to a maximum of 1000% or 2000%, based on the starting microorganism.
The term xe2x80x9cattenuationxe2x80x9d in this connection describes the reduction or elimination of the intracellular activity of one or more enzymes (proteins) in a microorganism which are coded by the corresponding DNA, for example by using a weak promoter or using a gene or allele which codes for a corresponding enzyme with a low activity or inactivates the corresponding gene or enzyme (protein), and optionally combining these measures.
By attenuation measures, the activity or concentration of the corresponding protein is in general reduced to 0 to 50%, 0 to 25%, 0 to 10% or 0 to 5% of the activity or concentration of the wild-type protein.
According to the invention, the system output of a fermentation unit producing L-threonine is increased by a procedure in which after a first fermentation step a portion of the fermentation broth obtained in this way remains in the production fermenter and serves as the inoculum for one or more further fermentation steps (batches).
According to the invention, 1 to 90 vol. %, preferably 1 to 50 vol. %, preferentially 1 to 25 vol. %, 1 to 20 vol. %, 1 to 15 vol. % or 1 to 10 vol. %, and particularly preferably 5 to 20 vol. %, 5 to 15 vol. % or 1 to 10 vol. % of the total volume of the fermentation broth remains in the fermentation tank.
The broth remaining in the fermentation tank is preferably topped up with a growth medium. After optionally  greater than 0 to not more than 10 hours, preferably after 1 to 10 hours, preferentially 2 to 10 hours and particularly preferably 3 to 7 hours a production medium is fed in. Alternatively, the components of this medium can also be fed in separately. After 20 to 72 hours, preferably 20 to 48 hours, the batch is ended and a portion of the fermentation broth, as described above, is separated off. A new fermentation stage is then optionally started with the remainder. The process can be repeated at least once, preferably approx. 2 to 6 times, depending on the stability of the strain used. Repetitions of approx. 2 to 8 times or 2 to 10 times or 2 to 4 times are also possible.
Appropriately stable strains which do not lose their production properties in the course of the process are particularly suitable for the process described.
The growth medium typically comprises sugars, such as e.g. glucose, starch hydrolysate, sucrose or molasses, as the source of carbon. Organic nitrogen-containing compounds, such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soya bean flour and urea, or inorganic compounds, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate, can be used as the source of nitrogen. The sources of nitrogen can be used individually or as a mixture. Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as the source of phosphorus.
The culture medium must furthermore comprise salts of metals, such as e.g. magnesium sulfate, manganese sulfate or iron sulfate, which are necessary for growth. Finally, essential growth substances, such as amino acids (e.g. homoserine) and vitamins (e.g. thiamine), are employed in addition to the above-mentioned substances. Antifoams, such as e.g. fatty acid polyglycol esters, can be employed to control the development of foam.
In general, the production medium comprises only one sugar, such as e.g. sucrose or glucose, and optionally an inorganic source of nitrogen, such as e.g. ammonium sulfate. Alternatively, these and other components can also be fed in separately.
During the growth or production phase, the temperature is established in a range from 29xc2x0 C. to 42xc2x0 C., preferably 33xc2x0 C. to 40xc2x0 C. Temperatures in a range from 27xc2x0 C. to 39xc2x0 C. are also possible. The fermentation can be carried out under normal pressure or optionally under increased pressure, preferably under an increased pressure of 0 to 1.5 bar. The oxygen partial pressure is regulated at 5 to 50%, preferably approx. 20% atmospheric saturation. Regulation of the pH to a pH of approx. 6 to 8, preferably 6.5 to 7.5, can be effected with 25% aqueous ammonia.
The process according to the invention is distinguished with respect to conventional processes above all by an increased space/time yield or productivity.
The present invention is explained in more detail in the following with the aid of embodiment examples.
The isolation of plasmid DNA from Escherichia coli and all techniques of restriction, Klenow and alkaline phosphatase treatment were carried out by the method of Sambrook et al. (Molecular Cloning. A laboratory manual (1989) Cold Spring Harbor Laboratory Press). Unless described otherwise, the transformation of Escherichia coli was carried out by the method of Chung et al. (Proceedings of the National Academy of Sciences of the United States of America USA (1989) 86: 2172-2175).